1. Field of the Invention
The present invention relates generally to the fields of sodium/bile acid cotransport systems in the ileum and kidney. Certain embodiments of the invention relate to the medically related fields of the control of blood cholesterol levels and treatments of diabetes, heart disease, liver disease and various digestive disorders. More particularly, the invention concerns the isolation and purification of bile acid cotransporter proteins and cDNA clones encoding the proteins and the use of these proteins and nucleic acids in therapeutic, preventative, genetic counseling and reagent screening applications.
2. Description of the Related Art
Bile acids are acidic sterols synthesized from cholesterol in the liver. Following synthesis, the bile acids are secreted into bile and enter the lumen of the small intestine, where they facilitate absorption of fat-soluble vitamins and cholesterol. Bile acids are then absorbed from the small intestine, returned to the liver via the portal circulation, and resecreted into bile. In the small intestine, bile acids are absorbed by both passive and active mechanisms (Dietschy, 1968). The active absorption of bile acids, first described by Lack and Weiner (1961), has been shown in man and experimental animals to be restricted to the ileum (Krag and Phillips, 1974; Schiff et al., 1972; Lack, 1979).
The first step in the active uptake of bile acids is mediated by a Na.sup.+ gradient-driven transporter located at the brush border (apical) membrane of the ileocyte (Wilson, 1981). Once inside the enterocyte, bile acids are transported across the cell to the basolateral membrane and secreted into the portal circulation via a Na.sup.+ -independent organic anion exchange system (Weinberg et al., 1986). The transport kinetics and specificity of this Na.sup.+ /bile acid cotransport system have been studied extensively using everted ileal gut sacs (Schiff et al., 1972; Lack, 1979), isolated ileocytes (Wilson et al., 1975; Schwenk et al., 1983), and ileal brush border membranes (Barnard and Ghishan, 1987; Kramer et al., 1992; Wilson and Treanor, 1979).
Although the mechanism of ileal bile acid transport has been characterized, the protein(s) responsible for this process have not been isolated and characterized. In an attempt to identify the proteins involved, photoaffinity studies have been performed using radiolabeled 7,7'-azo-derivatives of taurocholate with ileocytes and ileocyte membrane fractions (Kramer et. al., 1983; Lin et. al., 1990). These studies tentatively identified a brush border (apical) membrane protein of 99 kDa and basolateral membrane proteins of 54 and 59 kDa. More recently, lysylglycocholate-Sepharose affinity chromatography was used to isolate bile acid transporter-enriched ileal brush border membranes for polyclonal antibody production. In immunoblotting experiments, these antibodies detected a number of proteins including a 99 kDa protein in rat ileal brush border and kidney proximal tubule membranes. These antibodies also partially inhibited bile acid transport by isolated ileal brush border membranes (Gong et al., 1991). The tentative identification of a 90-99 kDa protein is also supported by chemical modification studies in rabbit ileum where agents that inhibited bile acid transport into ileal brush border membrane vesicles also blocked photoaffinity labeling of a 90 kDa protein (Kramer et al., 1992).
Notably lacking with the lysylglycocholate-Sepharose affinity chromatography and photoaffinity labeling studies was functional reconstitution of bile acid transport activity. Several candidate bile acid binding proteins previously identified by photoaffinity labeling have since been abandoned, illustrating the difficulties with this technique. For example, candidate 67 kDa and 43 kDa proteins were later shown to be albumin and actin, respectively (Fricker et al., 1982). Also, a candidate 54 kDa protein for the hepatic Na.sup.+ -independent multispecific anion transporter, has recently been shown to be Protein Disulfide Isomerase (Peter Meier, personal communication). The multispecific transporter has since been identified by expression cloning in Xenopus oocytes and shown to be a 75 kDa protein (Jacquemin et al., 1992).
Based on these observations, the 54 kDa protein identified in ileal basolateral membranes by photoaffinity labeling (Lin et al., 1988) may also be Protein Disulfide Isomerase. In addition, a candidate 49 kDa protein for the hepatic Na.sup.+ -dependent sinusoidal membrane bile acid transporter has been shown to be microsomal epoxide hydrolase (Von Dippe et al., 1993). Whereas the relationship between microsomal epoxide hydrolase and bile acid transport is still unclear, a candidate 40 kDa membrane glycoprotein with bile acid transport properties has independently been identified by expression cloning in Xenopus oocytes (Hagenbuch et al., 1991). Unfortunately, the identity of the ileal bile acid transporter has remained elusive because it has not been purified to homogeneity and functionally reconstituted or identified by cloning and expression.
The enterohepatic circulation of bile acids serves as a continuous link between the liver and small intestine. Disturbances in this cycling of bile acids have dramatic physiological consequences for both organ systems and for cholesterol homeostasis (Hofmann, 1989). This is illustrated by common disturbances of bile acid circulation including cholestasis and intestinal malabsorption. In addition to steatorrhea, bile acid malabsorption also has severe consequences for the colon, where excess bile acid results in watery diarrhea. Whereas the etiology of the bile acid malabsorption is clear in cases of ileal resection or Crohn's disease, specific defects in ileal bile acid transport or its regulation may also be responsible for some cases of chronic idiopathic diarrhea (Read et al., 1980; Heubi et al., 1981).
Bile acid malabsorption or disruption of the bile acid enterohepatic circulation stimulates de novo synthesis of bile acids in the liver. This results in an increased demand for cholesterol by the liver, which is compensated for by enhanced clearance of plasma LDL as well as increased hepatic cholesterol synthesis. If the malabsorption is significant, hepatic bile acid production may be unable to compensate for the loss, resulting in decreased intraluminal bile acid concentrations and a reduced ability to solubilize and absorb biliary and dietary cholesterol. This is the basis for the decreased plasma cholesterol levels and reduced morbidity from cardiovascular disease associated with ileal resection in the POSCH study (Program on the Surgical Control of Hyperlipidemias; Buchwald et al., 1990).
A less radical approach to treatment is the ingestion of polymeric bile acid sequestrants, such as cholestyramine and colestipol. Disruption of the bile acid enterohepatic circulation, especially in combination with HMG CoA reductase inhibitors, increases the number of hepatic LDL receptors and thereby decreases plasma LDL cholesterol levels (Brown and Goldstein, 1986). Despite gaps in the understanding of hepatic synthesis or intestinal conservation of bile acids, this drug regimen is widely used for the treatment of many forms of hypercholesterolemia (Goodman et al., 1988). Based on its remarkable substrate specificity, the ileal Na.sup.+ -dependent system represents an attractive target for treatment of hypercholesterolemia by interruption of the enterohepatic circulation with specific inhibitors of bile acid transport.
Therefore, bile acid transporter inhibitors are another immediate need in the art, which has not been met because the purified transporter is not available. Previous attempts to design inhibitors relied on the use of intact laboratory animals, isolated small intestine or kidney tissue, or isolated intestinal enterocytes. The use of laboratory animals instead of tissue culture cells or purified bile acid transporter enzyme made large scale drug screens prohibitively cumbersome and expensive. The number of compounds which could be assayed was also limited with small intestinal sections or isolated enterocytes, which are difficult to prepare and viable for only a period of hours.
Obviously, one would like to use the human ileal and renal bile acid transporters in these drug screens in the event that the animal model does not accurately mimic the human condition. However, human tissue is not readily available and very few human intestinal or kidney cell lines which express bile acid transporter activity exist. Currently, a human colon cancer cell line (CaCo.sub.2) has been discovered which expresses very small amounts of bile acid transporter activity. Unfortunately, this cell line requires special culture conditions and would be difficult to adapt to a high throughput assay format.
Therefore, there exists a need for a purified bile acid transporter to be used in the diagnosis and treatment of numerous human and animal conditions which are affected by this important system. In addition, a purified transporter would have utility in the screening of natural and man made products with possible pharmaceutical properties in the treatment of cholesterol related diseases and digestive and hepatic disorders.